Rapidly Growing Epstein-Barr Virus-Associated Pulmonary Lymphoma After Heart Transplantation

CASE REPORT

Rapidly growing Epstein-Barr virus-associated pulmonary lymphoma after heart transplantation

M. Schwend*, M. Tiemann**, H.H. Kreipe**, M.R. Parwaresch**, E.G. Kraatz+, G. Herrmann++, R.P. Spielmann$, J. Barth*

Rapidly growing Epstein-Barr virus-associated pulmonary lymphoma after heart trans- Dept of *Internal Medicine, **Hemato- plantation. M. Schwend, M. Tiemann, H.H. Kreipe, M.R. Parwaresch, E.G. Kraatz, G. pathology, +Cardiovascular Surgery, Herrmann, R.P. Spielmann, J. Barth. ERS Journals Ltd 1994. ++Cardiology, and $Radiographic Diagnostics, ABSTRACT: There is strong evidence to show an association of Epstein-Barr virus Christian-Albrechts-University of Kiel, Kiel, Germany. (EBV) infection with the development of post-transplant lymphoproliferative disease. We report the rapid development of a malignant lymphoma in a heart trans- Correspondence: J. Barth plant recipient, which occurred within less than eight weeks. I. Medizinische Universitätsklinik The diagnosis of this malignant high grade B-cell lymphoma was established by Schittenhelmstr. 12 open lung biopsy, and classified as centroblastic lymphoma of polymorphic subtype. D-24105 Kiel Immunohistochemically, the lymphoma showed reactivity with the B-cell markers Germany L-26 (CD20) and Ki-B5 and with the activation marker Ber-H2 (CD30). Furthermore, an expression of the bcl-2 oncoprotein was detected. Monoclonal JH gene rearrange- Keywords: Epstein-Barr virus ment was demonstrated by polymerase chain reaction (PCR), indicating monoclonal heart transplantation pulmonary lymphoma proliferation of B-blasts. Although serum EBV immunoglobulin M (IgM) antibodies were negative, the Received: July 1 1993 association to an EBV infection could be demonstrated by EBV immunostaining Accepted after revision November 5 1993 pattern which revealed an expression of the latent membrane protein (LMP) of EBV in the atypical blasts. The results give clear evidence of an EBV association of this rapidly growing lymphoma developed after heart transplantation. Eur Respir J., 1994, 7, 612Ð616.

The advances in immunosuppression in the past decade We report the case of a rapidly growing pulmonary have greatly improved the feasibility of transplantation lymphoma classified by immunohistochemical methods medicine. Whereas successful immunosuppression pre- and polymerase chain reaction (PCR) analysis. vents rejection of the transplant and maintains the function of the organ, it also is the basis of transplantation associated complications, which mostly affect the lung Case report [1]. In permanent contact with the external milieu, the lung is especially prone to infections in immunosup- A 51 year old male with a New York Heart Association pressed patients [2]. Furthermore, an increase of pri- class IV dilated cardiomyopathy underwent orthotopic mary and secondary malignancies of the lung occurs in heart transplantation in September 1990. Postoperatively, transplantation patients, which may cause considerable immunosuppressive therapy was started with antithy- differential diagnostic difficulties due to rapid tumour mocyte globulin, followed by a triple combination of growth or unusual clinical and radiographic manifesta- cyclosporin A, azathioprine and methylprednisolone. An tions [1, 2]. Although the incidence of common carci- endomyocardial biopsy performed eight weeks after trans- nomas is not increased, there is a considerable increase plantation revealed no histological evidence of allograft of lymphomas occurring after transplantation [3, 4]. The rejection, and a chest roentgenogram displayed no abnor- Epstein-Barr virus (EBV) plays a crucial role in the patho- malities of the lung structure. After another two months, genesis of post-transplantational lymphoproliferative dis- myocardial biopsy still failed to display any evidence of ease [5Ð8]. a rejection, whereas a subsequent chest X-ray revealed Before the introduction of modern immunohisto- symmetrical, patchy infiltrates in both lungs, mainly occur- chemical and molecular biological techniques with ring in the middle and lower fields (fig. 1). The patient appropriate monoclonal antibodies and gene probes, it was free of symptoms; specifically, he had no history was difficult to characterize the malignant tumour and of fever, weight loss, nocturnal sweat, or dyspnoea. to support the proposed association of post-transplant Physical examination demonstrated only slight high lymphomas to an EBV infection. pitched crackles over both lungs. The erythrocyte EBV-ASSOCIATED PULMONARY LYMPHOMA POST-TRANSPLANT 613

Fig. 1. Ð Chest X-ray after a routine myocardial biopsy, accidentally Fig. 2. Ð Computer tomography of the thorax showing multiple pul- demonstrating infiltrates over both lungs. monary nodules with preference to the middle fields of both lungs. sedimentation rate (ESR) was 50/77 mm, haemoglobin Open lung biopsy demonstrated a high grade malignant 11.2 gádl-1, urea 92.6 mgádl-1, and creatinine 1.69 mgádl-1. B-cell lymphoma, which was classified immunohisto- Lactate dehydrogenase was increased at 274 Uál-1 (nor- chemically as a centroblastic lymphoma of polymorphic mal <240 Uál-1). Normal values were found for other subtype (high grade germinal centre lymphoma). A serum enzymes, serum electrophoresis and the tumour monoclonal light chain expression was not found. markers alpha-fetoprotein (AFP), carbohydrate antigenic Azathioprine therapy was stopped and the dosage of determinant 19-9 (CA 19-9) and carcinoembryonic anti- cyclosporin A was decreased. Subsequently, combi- gen (CEA). Paraproteins were not detectable in serum nation chemotherapy with cyclophosphamide, doxoru- and urine. Sputum and gastric juice samples were free bicin, vincristine and prednisolone was initiated. The from tuberculosis bacteria. Antibody screening was nega- patient died unexpectedly because of cardiorespiratory tive for EBV immunoglobulin M (IgM) and showed no failure shortly after the first therapy cycle was com- evidence of a fresh EBV infection; however, it revealed pleted. Permission for autopsy was not obtained. a past primary EBV infection. From cultures and serological tests there was no evidence of a current infection with other pneumotropic viruses, Candida, Aspergillus, Immunohistochemical and molecular analysis Legionella pneumophila or Mycoplasma. A computed tomography (CT)-scan of the thorax visualized multiple pulmonary nodules, mainly in the middle fields (fig. 2). Material and methods Lung function tests revealed a restrictive pattern, with a decreased vital capacity of 3.2 l (predicted 4.47 l) and Immunohistochemistry of lymphoma tissue was per- a decreased arterial oxygen tension (PaO2) of 54 mmHg formed with the alkaline phosphatase and anti-alkaline (7.2 kPa). The single-breath diffusing capacity of the phosphatase (APAAP) staining system, according to lungs for carbon monoxide (DLCO) was reduced to 3.67 CORDELL et al. [9], with a monoclonal antibody against mmolámin-1ákPa-1 (46% predicted). Fibreoptic broncho- EBV latent membrane antigen (LMP-1) encoded by the scopy showed a low grade inflammation without circum- BNLF1 gene of the EBV virus, which is expressed in script alterations of the bronchial system. Transbronchial EBV-infected cells (Dako, Hamburg, FRG) [10]. Further- biopsies revealed an interstitial fibrosis without evidence more, staining with the bcl-2 oncoprotein antibody (Dako, of a specific pneumonia or malignancy. Hamburg, FRG) was performed, marking an antigen The bronchoalveolar lavage fluid contained 64% macro- which is expressed in germinal centre cell lymphoma phages, 35% lymphocytes and 1% granulocytes. The with the 14;18 translocation [11]. The B-cell nature of analysis of lymphocyte subpopulations displayed 89% the lymphoma was demonstrated by staining with the CD3, 44% CD4, 48% CD8, and 4% CD19 positive B- monoclonal B-cell antibodies Ki-B5 [12] (Parwaresch, lymphocytes. The CD4/CD8 ratio was 0.92. Immuno- Kiel, FRG) and L-26 (CD20, Dako, Hamburg, FRG) staining for pneumocystis carinii and cytomegalovirus [13]. An anti CD3 polyclonal antibody was also obtained (CMV) early antigen was negative. Further investiga- (Dako, Hamburg, FRG), whereas the Ber-H2 monoclonal tions, including abdominal ultrasound and bone marrow antibody, which recognizes an activation antigen (CD30), biopsies, revealed no pathological findings leading to a also expressed in EBV infected cells, was obtained by diagnosis. Stein (Berlin, FRG) [14]. Deoxyribonucleic acid (DNA) 614 M. SCHWEND ET AL. was studied by polymerase chain reaction (PCR) tech- Table 1. Ð Immunoreactivity pattern of lymphoma cells nique for IgH rearrangement. DNA was extracted from paraffin blocks, as described by WAN et al. [15]. After Antibody Cluster Immunoreactivity Source PCR, mineral oil was removed and samples were extracted with chloroform. Five microlitres of each sam- L-26 CD20 + Dako, Hamburg, FRG ple was loaded onto a polyacrylamide gel for 100 min Ki-B5 - + Parwaresch, Kiel, FRG Ber-H2 CD30 + Stein, Berlin, FRG at 400 V over a temperature gradient of T1 10¡C and T2 Anti-CD3 CD3 - Dako, Hamburg, FRG 60¡C. After electrophoresis, fractions were stained using bcl-2 - + Dako, Hamburg, FRG a silver-staining procedure, according to ROSENBAUM and LMP - + Dako, Hamburg, FRG RIESNER [16]. LMP: latent membrane protein. Results and interpretation The regular B-cell antigens, Ki-B 5 and L-26, could Immunohistochemical investigations revealed an expres- be demonstrated on all atypical blasts, including small sion of the activation antigen CD30 and of the bcl-2 centroblasts. The immunostaining pattern of lymphoma oncoprotein in a minority of cells (fig. 3). Bcl-2 expres- cells is summarized in table 1. After PCR with primers sion is associated with the production of an inner mito- specific for heavy chain consensus sequences [15], using chondrial membrane protein that blocks programmed temperature gradient gel electrophoresis, one mono- cell death [17], which results in a growth advantage of clonal band was detected, which has to be interpreted as bcl-2 positive lymphoma cells. monoclonal proliferation of B-blasts [15]. Additionally, LMP could be detected in larger, Hodgkin- cell-like immunoblasts (fig. 4), indicating the EBV- association of the lymphoma [10]. Discussion Pulmonary infections are the most frequent and impor- tant complications after heart transplantation. According to recent studies, they occur with a frequency of 24Ð40% [2, 18]. They normally develop within the first 3Ð4 months after transplantation, and they often show a lethal course at this stage [19]. Even after this period, pulmonary infections still constitute a major complication of heart transplantation. In this particular case, the radiographic alterations developed within a few weeks, so that a possible infectious cause had to be taken into consi- deration, even if no clinical symptoms were detectable. The clinical course and the diagnostic procedures, including BAL, transbronchial biopsies and serological findings, ruled out a current infectious disease, but they did Fig. 3. Ð Alkaline phosphatase and anti-alkaline phosphatase (APAAP) not allow a definitive diagnosis. The CT-scan of the immunostaining of lymphoma cells with a monoclonal antibody against lungs pointed to a malignant process; therefore, an open bcl-2 oncoprotein (black arrow), which is expressed in germinal cen- lung biopsy was performed and the diagnosis of this high tre cell lymphoma with the 14;18 translocation. (Scale bar = 10 µm). grade malignant B-cell lymphoma was established. Organ recipients after allotransplantation carry a stron- gly increased risk of malignancy [20Ð22]. Whereas, the incidence of common carcinomas like bronchial, breast, colon, or prostate carcinoma is not increased, there is an increase of particular malignancies, such as the hepato- biliary carcinoma, the Kaposi-sarcoma, and, most of all, the lymphomas. According to recent studies, the high- est rate of lymphomas is found in heart and heart/lung transplanted patients, with a 3.5Ð7% risk of developing a lymphoproliferative disease after transplantation [3, 4]. These lymphomas are frequently (about 30%) located in the lungs, which is an uncommon location otherwise. The risk of a post-transplantation lymphoproliferative disorder increases with the intensification of the immunosuppressive therapy [23]. In this case, the patient ini- Fig. 4. Ð APAAP immunostaining of lymphoma cells with a mono- tially received antithymocyte globulin, followed by a clonal antibody against latent membrane protein (LMP) expressed in Epstein-Barr virus (EBV)-infected cells. An apoptosis of a LMP- long-term triple combination of cyclosporin A, azathio- negative cell is shown (black arrow) as well as a mitotic cell (white prine and methylprednisolone. If cyclosporin A is a part arrow). (Scale bar = 10 µm). of the immunosuppressive regime, lymphomas are more EBV-ASSOCIATED PULMONARY LYMPHOMA POST-TRANSPLANT 615 frequent and they occur earlier in the post-transplantation involved the use of monoclonal anti-B cell antibodies period. The short time of 10 months between trans- and showed good results. plantation and clinical appearance of this lymphoma cor- responds to the findings of epidemiological studies, which showed that lymphomas most frequently occur within a References few months up to a year after initiating the immuno- 1. Ettinger NA, Trulock EP. Pulmonary considerations of suppressive therapy with cyclosporin [3, 22]. In con- organ transplantation. Part 3. 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